In recent years, it has become common in clinical laboratories to test for the level of cholesterol in blood serum samples. The measured levels of cholesterol in the blood are referred to as total serum cholesterol, and include all those cholesterol compounds which are present in the blood such as cholesterol and its derivatives dihydrocholesterol and 7-dihydrocholesterol, whether present in their free form or in the form of esters with the fatty acids normally present in the blood. There are a number of methods which can be utilized for the determination of total cholesterol in biological fluids. In accordance with the method of Abell et al, the serum is treated with alcoholic potassium hydroxide to liberate the cholesterol from its lipoprotein complexes and to saponify the natural cholesterol esters. The saponified cholesterol is extracted into a measured volume of petroleum ether and then an aliquot is subjected to color reaction utilizing a modified Libermann-Burchard reagent. Reference can be made to the journal "STANDARDIZED METHODS OF CLINICAL CHEMISTRY", vol. 2, pages 26 etc. (1958) by L. L. Abell et al. The optical density of each sample is read against a blank in a photoelectric colorimeter. The level of cholesterol equivalent to the optical density is calculated by comparing the optical density to that of a standard containing a known amount of cholesterol.
For most serum clinical chemistry procedures, it is desirable to use one serum based reference control for a variety of tests. However, cholesterol, per se, is not soluble in biological fluids such as serum. To overcome such deficiency, the prior art has attempted to solubilize the cholesterol by forming organic salts thereof. See, for example, U.S. Pat. No. 3,859,047 to Klein. However, the level of solubility achieved is insufficient to provide sufficiently high concentrations for all desired uses. For example, even with the use of a surfactant such as Triton X-100 (polyethyleneglycol ether of monoisoocytyl phenol, by Rohm & Haas, Inc., Philadelphia, Pa.) a useful concentration of less than 0.1 gram per deciliter is provided. The sample becomes turbid at significantly higher concentrations, and since a colorimetric procedure is utilized, gross errors can be introduced.
More recently, a method for cholesterol analysis employing the enzyme cholesterase has been developed and has been increasingly used in analytical laboratories. Accordingly, it will be desirable for a cholesterol standard to be useful not only in the saponification chemistry of the Abell et al method but also in the enzymatic procedures.
The present invention provides serum-soluble cholesterol compounds and methods for their preparation, which can be lyophilized and added in high concentration to blood serum. A turbid-free clinical chemistry control is thus provided having a high cholesterol value and which can be assayed by the standard or enzymatic procedures. Concentration levels of up to 1 gram of cholesterol equivalent per deciliter are easily prepared. Furthermore, the cholesterol compounds of this invention do not interfere with other analytical tests making them highly useful in a multiple serum control.
More specifically, the present cholesterol compounds are defined by certain esters of cholesterol reactively combined with a solubilizing agent selected from the group consisting of peptides, proteins, water-soluble polycarboxylic acids, organic and inorganic water-soluble salts of said acids, and cis-vicinal water-soluble polysaccharides. With regard to the peptides and proteins, reaction is with the amino groups derived therefrom (i.e., as a part thereof). The ester can be: (A) an acid ester of cholesterol, in which the acid group has at least six carbon atoms in its longest chain (including the acid carbon atoms), reactable with the amine component of the peptide or protein amino residue; or (B) an amino ester of cholesterol reactable (1) directly with the acid portion of the peptide or protein amino residue or with the polycarboxylic acid, (2) indirectly through a phosgene or thiophosgene coupling agent with the amine component of the peptide or protein amino residue, or (3) indirectly through a cyanogen halide with a polysaccharide.